For the evaluation of the Reactive Oxygen Species (ROS) produced and released in vitro, cells were seeded as described before for the MTT test; the analysis were performed 24, 48, 72 and 96 hours after the addition of clusters in culture medium; a ROS-Glo™ H2O2 Assay (Promega) was used, following the producer protocol. Unstained cells are reported as negative control. The Non-Lytic assay was applied and the relative luminescence units were measured by a plate reader (GloMax Discover, Promega). For solution studies, triplicate samples of metallic clusters in water (7 μM) were tested using ROS-Glo™ H2O2 Assay (Promega), following the manufacturers’ protocol. An opaque white 96 wells plate was loaded with 50 μl of each sample dispersed in H2O2 (5 mM) saturated water, followed by the incubation with H2O2 substrate solution and ROS-Glo detection solution. After 20 min incubation at room temperature, the luminescence was measure by GloMax Discover (Promega) plate reader. The luminescence was recorded continuously every 15 minutes for 5 hours.
Ros glo h2o2 assay
Manufactured by Promega
Sourced in United States, United Kingdom, Germany, France
The ROS-Glo™ H2O2 Assay is a luminescent assay designed to detect and quantify hydrogen peroxide (H2O2) in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Glomax multi detection system, by Promega (5 mentions)
Caspase glo 3 7 assay, by Promega (5 mentions)
Gsh gssg glo assay, by Promega (5 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Nunclon 96 flat white plate, by Thermo Fisher Scientific (3 mentions)
Mitochondrial toxglo assay, by Promega (3 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (3 mentions)
Catalase, by Merck Group (2 mentions)
Menadione, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Celltiter glo assay, by Promega (2 mentions)
Infinite m1000, by Tecan (2 mentions)
Fluostar omega, by BMG Labtech (2 mentions)
Reliaprep rna cell miniprep system, by Promega (2 mentions)
Sigma fast bcip nbt reagent, by Merck Group (2 mentions)
Polyclonal rabbit anti β tubulin antibody, by Cell Signaling Technology (2 mentions)
Caspase glo 1 inflammasome assay, by Promega (2 mentions)
148 protocols using ros glo h2o2 assay
1
Quantifying ROS Production In Vitro
2
Multiparametric Cytotoxicity Evaluation
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The analyses were done 24 h after the exposure to allow the cells to respond to the exposure. In order to analyze cell viability and oxidative stress in the same cell, two cell-based assays were combined. The oxidative stress was analyzed first, using the ROS-Glo™ H2O2 Assay (Promega, Madison, WI, USA). Afterwards, cell viability was measured with the CellTiter-Blue® Assay (Promega).
For the ROS-Glo™ H2O2 Assay, AEGM medium (200 µL) and H2O2 substrate solution (50 µL) were added on the surface of the cells. After 3 h incubation at 37 °C/5% CO2, 75 µL of the solution was transferred into a white 96 well plate. Detection solution (75 µL) was added and after 20 min incubation at room temperature, the relative luminescence was measured.
In order to measure the cell viability in the same cell culture insert, the remaining medium was removed from the cells, and 300 µL AEGM medium as well as 60 µL CellTiter-Blue® solution were added. The cultures were then incubated for 2 h at 37 °C/5% CO2. Afterwards, 100 mL of the solution were pipetted into a black 96 well plate to measure the fluorescence at 544Ex/590Em nm.
For the ROS-Glo™ H2O2 Assay, AEGM medium (200 µL) and H2O2 substrate solution (50 µL) were added on the surface of the cells. After 3 h incubation at 37 °C/5% CO2, 75 µL of the solution was transferred into a white 96 well plate. Detection solution (75 µL) was added and after 20 min incubation at room temperature, the relative luminescence was measured.
In order to measure the cell viability in the same cell culture insert, the remaining medium was removed from the cells, and 300 µL AEGM medium as well as 60 µL CellTiter-Blue® solution were added. The cultures were then incubated for 2 h at 37 °C/5% CO2. Afterwards, 100 mL of the solution were pipetted into a black 96 well plate to measure the fluorescence at 544Ex/590Em nm.
3
Menadione-Induced ROS Quantification
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To measure ROS induced by menadione, we used ROS-Glo™ H2O2 assay (Promega, G8820). PC6 cells were grown overnight on an opaque white 96 well plate. On the next day, the cells were treated with menadione (different concentrations) alone or in combination with cell-permeable reduced glutathione ethyl ester (GSH-MEE) and subjected to ROS-Glo™ H2O2 assay (Promega). Briefly, the H2O2 substrate solution were added, bringing the final volume to 100 μl per well. The plate was incubated at 37 °C in a 5% CO2 incubator. After 4 h incubation, 100 μl of the ROS-Glo detection solution was added to each well and further incubated the plate for 30 min at room temperature. The luminescence was recorded using the Glomax Multi Detection System (Promega).
4
Dual Assay for Cellular Stress and Viability
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The analyses were done 24 h after the exposure, allowing the cells to respond to the exposure. In order to analyze cell viability and oxidative stress in the same cell, two cell-based assays were combined. The oxidative stress was analyzed first, using the ROS-Glo™ H2O2 Assay (Promega, Madison, WI, USA). Afterwards, cell viability was measured using the CellTiter-Blue® Assay (Promega).
For the ROS-Glo™ H2O2 Assay, AEGM medium (200 µL) and H2O2 substrate solution (50 µL) were added on the surface of the cells. After 3 h incubation at 37 °C/5% CO2, 75 µL of the solution was transferred into a white 96-well plate. Detection solution (75 µL) was added and after 20 min incubation at room temperature, the relative luminescence was measured.
In order to measure the cell viability in the same cell culture insert, the remaining medium was removed from the cells, and 300 µL AEGM medium as well as 60 µL CellTiter-Blue® solution were added. The cultures were then incubated for 2 h at 37 °C/5% CO2. Afterwards, 100 mL of the solution were pipetted into a black 96-well plate to measure the fluorescence at 544Ex/590Em nm.
For the ROS-Glo™ H2O2 Assay, AEGM medium (200 µL) and H2O2 substrate solution (50 µL) were added on the surface of the cells. After 3 h incubation at 37 °C/5% CO2, 75 µL of the solution was transferred into a white 96-well plate. Detection solution (75 µL) was added and after 20 min incubation at room temperature, the relative luminescence was measured.
In order to measure the cell viability in the same cell culture insert, the remaining medium was removed from the cells, and 300 µL AEGM medium as well as 60 µL CellTiter-Blue® solution were added. The cultures were then incubated for 2 h at 37 °C/5% CO2. Afterwards, 100 mL of the solution were pipetted into a black 96-well plate to measure the fluorescence at 544Ex/590Em nm.
5
Oxidative Stress and Cell Viability Assays
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All materials were reagent grade. Minimal essential media (MEM) Glutamax, 100X antibiotic/antimycotic, heat-inactivated FBS, 100 mM sodium pyruvate, and 0.25% Trypsin–EDTA were from Life Technologies (Grand Island, NY, USA). Alamethicin was from Cayman Chemical (Ann Arbor, MI, USA). DMSO, menadione, eseroline (fumarate salt), benserazide, pyrogallol, and catalase were from Sigma-Aldrich (St. Louis, MO, USA). CellTiter-Glo® and ROS-Glo™ H2O2 Assays were from Promega Corp. (Madison, WI, USA).
6
ROS-Glo H2O2 Assay Protocol
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ROS-Glo H2O2 assays were performed according to the manufacturer’s protocol (Promega). Cells were incubated with H2O2 substrate solution for 6 h. The media was then incubated with the ROS-Glo detection solution containing d -Cysteine and the signal enhancer solution for 20 min at room temperature. Luminescence intensities from the mixture were measured by the GloMaxMulti Detection System (Promega).
7
iPSC-derived Cardiomyocyte Assays
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Patient‐specific iPSC‐derived cardiomyocytes were seeded on 96‐well plates subjected to plate‐based assays after drug treatment using SpectraMax 340PC384, SpectraMax L, and FlexStation 3 microplate readers. CYTO‐ID autophagy detection kits 2.0 (Enzo Life Sciences) were used for autophagy assays, ROS‐Glo H2O2 assays (Promega) were used for ROS detection, JC‐1 assays (Enzo Life Sciences) were used for mitochondrial membrane potential measurement, and Cell Titer Glo 2.0 assays (Promega) were used for ATP content, in accordance with the manufacturers' instructions.
8
Quantifying Oxidative Stress via H2O2 Assay
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The oxidative stress was analyzed using the ROS-Glo™ H2O2 Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The ROS-Glo™ H2O2 Assay is a bioluminescent assay that measures the level of hydrogen peroxide (H2O2), a reactive oxygen species (ROS), directly in cell culture or in defined enzyme reactions. Briefly, 2 × 104 cells were seeded in 96-well plates and cultured in a complete DMEM medium. The studied T (10–60 µg/mL), DT1 (0.01–0.08 µg/mL), DT2 (500–2000 µg/mL) were then added, and the cells were incubated at 37 °C in a 5% CO2 atmosphere for 24 h. H2O2 substrate solution was added for 6 h to generate a luciferin precursor. The addition of ROS-Glo™ Detection Solution (20 min) converts the precursor to luciferin and provides Ultra-Glo™ Recombinant Luciferase to produce a light signal that is proportional to the level of H2O2 present in the sample. The relative luminescence was measured using a GloMax®® Discover Microplate Reader.
9
Quantifying Cellular Hydrogen Peroxide
10
Proteasome and Caspase Activity Assay
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Cells were seeded in a white 96-well plate at an 8000 cells/well density and incubated overnight for attachment. Cells were treated with either DMSO control, MG-132 (1 µM), staurosporin (0.1 μM) or Foldlin (at IC50 concentration) for a total incubation time of 8 h (proteasome) or 16 h (caspase test). Proteasomal activity and caspase activity were monitored according to the manufacturer’s guidelines (ROS-Glo H2O2 assay, Promega, Leiden, The Netherlands; Proteasome-Glo, Promega, Leiden, The Netherlands, caspase-GLO 3/7 assay, Promega, Leiden, The Netherlands).
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